PROJECT SUMMARY The recent emergence of high-throughput DNA sequencing technologies [1-6] and their application to transcriptomics (by sequencing complementary DNA (cDNA)) [7-12] have resulted in an even more complicated view of the transcriptome and its regulation, demonstrating our limited knowledge of transcription. DNA sequencing technologies have eliminated some of the technical challenges posed by hybridization-based microarray strategies, but several fundamental shortcomings still remain [13-18] that prevent us from understanding the ?true? nature of transcriptomes and from identifying the 1?2 fold transcription changes that can distinguish a disease-associated physiological process from a normal one [19, 20]. To respond to this need, SeqLL, LLC (Woburn, MA) is proposing a Phase I SBIR project to develop, optimize, and demonstrate the feasibility of a direct RNA sequencing technology to capture whole-transcriptome information from the RNA isolated from a single cell. This work builds on encouraging preliminary data demonstrating the feasibility of a direct RNA sequencing technology based on a single-molecule sequencing platform; however, existing challenges must be overcome for this technology to be a viable and reliable commercial option (including a reduced read length compared to DNA sequencing, a lower yield compared to a similar input of DNA, and is less reproducible in lower-order species). To achieve this goal, the team proposes three specific aims: Specific Aim 1. To design, synthesize, and screen multi-color reversible terminators optimized for the sequencing of RNA molecules. This aim will use chemical synthesis methods to generate reversible terminator (RT) analogs that are well suited to direct RNA sequencing. These RTs will be ?third generation? (based on innovative iterations of these molecules by SeqLL), fluorescently labeled, and have a variety of inhibitory moieties that will be tested for improved sequencing performance. Specific Aim 2. Optimize chemical and reagents for use with high-throughput single-molecule direct RNA sequencing to improve base stacking, sequencing yield, read length, and accuracy. This aim will optimize imaging buffer, generate mechanisms for cleave/capping, and to test alternative modifications that should allow for significant improvements in direct RNA sequencing base stacking, sequencing yield, read length, and accuracy. Specific Aim 3. Whole-transcriptome sequencing of single cell RNA without amplification. This aim will seek to efficiently perform whole-transcriptome sequencing of RNA derived from a single cell, without amplification. Single-cell preparations will be obtained from our collaborators at the Bernstein lab (Harvard Medical School). This work will report upon methods and the sequencing performance of using the optimized methods identified in Aims 1 and 2 to perform single-cell direct RNA sequencing of the whole transcriptome.